Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates

Maya Tanase, Valerio Zolla, Cristina C. Clement, Francesco Borghi, Aleksandra M. Urbanska, Jose Antonio Rodriguez-Navarro, Barbara Roda, Andrea Zattoni, Pierluigi Reschiglian, Ana Maria Cuervo, Laura Santambrogio

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Herein we describe a protocol that uses hollow-fiber flow field-flow fractionation (FFF) coupled with multiangle light scattering (MALS) for hydrodynamic size-based separation and characterization of complex protein aggregates. The fractionation method, which requires 1.5 h to run, was successfully modified from the analysis of protein aggregates, as found in simple protein mixtures, to complex aggregates, as found in total cell lysates. In contrast to other related methods (filter assay, analytical ultracentrifugation, gel electrophoresis and size-exclusion chromatography), hollow-fiber flow FFF coupled with MALS allows a flow-based fractionation of highly purified protein aggregates and simultaneous measurement of their molecular weight, r.m.s. radius and molecular conformation (e.g., round, rod-shaped, compact or relaxed). The polyethersulfone hollow fibers used, which have a 0.8-mm inner diameter, allow separation of as little as 20 μg of total cell lysates. In addition, the ability to run the samples in different denaturing and nondenaturing buffer allows defining true aggregates from artifacts, which can form during sample preparation. The protocol was set up using Paraquat-induced carbonylation, a model that induces protein aggregation in cultured cells. This technique will advance the biochemical, proteomic and biophysical characterization of molecular-weight aggregates associated with protein mutations, as found in many CNS degenerative diseases, or chronic oxidative stress, as found in aging, and chronic metabolic and inflammatory conditions.

Original languageEnglish (US)
Pages (from-to)134-148
Number of pages15
JournalNature Protocols
Volume10
Issue number1
DOIs
StatePublished - Jan 1 2015

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Hydrodynamics
Fractionation
Field Flow Fractionation
Molecular Weight
Light scattering
Molecular Conformation
Light
Fibers
Flow fields
Proteins
Paraquat
Molecular weight
Central Nervous System Diseases
Ultracentrifugation
Complex Mixtures
Carbonylation
Proteomics
Artifacts
Oxidative stress
Gel Chromatography

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Tanase, M., Zolla, V., Clement, C. C., Borghi, F., Urbanska, A. M., Rodriguez-Navarro, J. A., ... Santambrogio, L. (2015). Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates. Nature Protocols, 10(1), 134-148. https://doi.org/10.1038/nprot.2015.009

Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates. / Tanase, Maya; Zolla, Valerio; Clement, Cristina C.; Borghi, Francesco; Urbanska, Aleksandra M.; Rodriguez-Navarro, Jose Antonio; Roda, Barbara; Zattoni, Andrea; Reschiglian, Pierluigi; Cuervo, Ana Maria; Santambrogio, Laura.

In: Nature Protocols, Vol. 10, No. 1, 01.01.2015, p. 134-148.

Research output: Contribution to journalArticle

Tanase, M, Zolla, V, Clement, CC, Borghi, F, Urbanska, AM, Rodriguez-Navarro, JA, Roda, B, Zattoni, A, Reschiglian, P, Cuervo, AM & Santambrogio, L 2015, 'Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates', Nature Protocols, vol. 10, no. 1, pp. 134-148. https://doi.org/10.1038/nprot.2015.009
Tanase M, Zolla V, Clement CC, Borghi F, Urbanska AM, Rodriguez-Navarro JA et al. Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates. Nature Protocols. 2015 Jan 1;10(1):134-148. https://doi.org/10.1038/nprot.2015.009
Tanase, Maya ; Zolla, Valerio ; Clement, Cristina C. ; Borghi, Francesco ; Urbanska, Aleksandra M. ; Rodriguez-Navarro, Jose Antonio ; Roda, Barbara ; Zattoni, Andrea ; Reschiglian, Pierluigi ; Cuervo, Ana Maria ; Santambrogio, Laura. / Hydrodynamic size-based separation and characterization of protein aggregates from total cell lysates. In: Nature Protocols. 2015 ; Vol. 10, No. 1. pp. 134-148.
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